Kohno H, Kohno T, Sakoda I, Ishikawa E
Fuji Research Laboratories, Kyowa Medex Co., Ltd., Shizuoka, Japan.
J Clin Lab Anal. 1990;4(5):355-62. doi: 10.1002/jcla.1860040508.
A novel and sensitive enzyme immunoassay (immune-complex-transfer enzyme immunoassay) for antihuman T cell leukemia virus type 1 IgG (anti-HTLV-1 IgG) in human serum using recombinant gag(14-139)-env-(197-295) hybrid protein is described. Anti-HTLV-1 IgG in test serum was reacted with dinitrophenyl biotinyl bovine serum albumin-recombinant gag-env hybrid protein conjugate. The complex formed was trapped onto polystyrene balls coated with affinity-purified antidinitrophenyl group IgG. After washing to eliminate nonspecific IgG in the test serum, the complex was eluted from the polystyrene balls with dinitrophenyl-L-lysine and transferred to polystyrene balls coated with streptavidin. After washing, anti-HTLV-1 IgG in the complex trapped onto the streptavidin-coated polystyrene balls was reacted with antihuman IgG gamma-chain Fab'-peroxidase conjugate. Peroxidase activity bound to the streptavidin-coated polystyrene balls was assayed by fluorometry. By transfer of the complex, the nonspecific binding of nonspecific human IgG was considerably reduced, and the detection limit of anti-HTLV-1 IgG in serum was lowered 30-300-fold compared with that by Western blotting, gelatin particle agglutination, and the conventional enzyme immunoassay, in which a recombinant gag-env hybrid protein-coated polystyrene ball was incubated with test serum and, after washing, with antihuman IgG gamma-chain Fab'-peroxidase conjugate. Usefulness of the immune-complex-transfer enzyme immunoassay was demonstrated using 271 serum samples.
描述了一种新型灵敏的酶免疫测定法（免疫复合物转移酶免疫测定法），用于检测人血清中抗人类T细胞白血病病毒1型IgG（抗-HTLV-1 IgG），该方法使用重组gag(14-139)-env-(197-295)杂交蛋白。检测血清中的抗-HTLV-1 IgG与二硝基苯基生物素化牛血清白蛋白-重组gag-env杂交蛋白偶联物反应。形成的复合物被捕获到包被有亲和纯化的抗二硝基苯基IgG的聚苯乙烯球上。洗涤以去除检测血清中的非特异性IgG后，用二硝基苯基-L-赖氨酸从聚苯乙烯球上洗脱复合物，并转移到包被有链霉亲和素的聚苯乙烯球上。洗涤后，捕获在包被有链霉亲和素的聚苯乙烯球上的复合物中的抗-HTLV-1 IgG与抗人IgGγ链Fab'-过氧化物酶偶联物反应。通过荧光法测定结合在包被有链霉亲和素的聚苯乙烯球上的过氧化物酶活性。通过复合物的转移，非特异性人IgG的非特异性结合显著降低，与蛋白质印迹法、明胶颗粒凝集法和传统酶免疫测定法相比，血清中抗-HTLV-1 IgG的检测限降低了30-300倍，传统酶免疫测定法是将包被有重组gag-env杂交蛋白的聚苯乙烯球与检测血清孵育，洗涤后再与抗人IgGγ链Fab'-过氧化物酶偶联物孵育。使用271份血清样本证明了免疫复合物转移酶免疫测定法的实用性。
